NSAIDs interactions with membranes: a biophysical approach

This work focuses on the interaction of four representative NSAIDs (nimesulide, indomethacin, meloxicam, and piroxicam) with different membrane models (liposomes, monolayers, and supported lipid bilayers), at different pH values, that mimic the pH conditions of normal (pH 7.4) and inflamed cells (pH 5.0). All models are composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) which is a representative phospholipid of most cellular membranes. Several biophysical techniques were employed: Fluorescence steady-state anisotropy to study the effects of NSAIDs in membrane microviscosity and thus to assess the main phase transition of DPPC, surface pressure-area isotherms to evaluate the adsorption and penetration of NSAIDs into the membrane, IRRAS to acquire structural information of DPPC monolayers upon interaction with the drugs, and AFM to study the changes in surface topography of the lipid bilayers caused by the interaction with NSAIDs. The NSAIDs show pronounced interactions with the lipid membranes at both physiological and inflammatory conditions. Liposomes, monolayers, and supported lipid bilayers experiments allow the conclusion that the pH of the medium is an essential parameter when evaluating drug-membrane interactions, because it conditions the structure of the membrane and the ionization state of NSAIDs, thereby influencing the interactions between these drugs and the lipid membranes. The applied models and techniques provided detailed information about different aspects of the drug-membrane interaction offering valuable information to understand the effect of these drugs on their target membrane-associated enzymes and their side effects at the gastrointestinal level.     

Interaction of a laminin derived peptide with phosphatidyl choline/phosphatidyl glycerol

The physicochemical interactions between an active peptide sequence derived from laminin and phospholipids have been studied. The main aim is to determine the suitability of this peptide fragment to be linked to liposome's with the purpose to develop targeting devices. Results indicate that this peptide is able to insert in lipid monolayers and also to form monomolecular layers at an air/water interface. Besides, miscibility studies carried out through compression isotherms of mixed monolayers [dipalmitoyl phosphatidylcholine (DPPC)/peptide], indicate a strong interaction at 60-80% DPPC molar composition. Studies carried out with lipid bilayers indicate that the interaction is restricted to the external face of the vesicles. Moreover, the presence of this peptide in the incubation media promotes a low level of carboxyfluorescein (CF) leakage and no fusion of vesicles. These results indicate that the association of this sequence to vesicles do not produce damage of the bilayer and can be used as potential targeting vector.     

Membrane interactions of host-defense peptides studied in model systems

Host-defense, antibiotic peptides are believed to generate their cytolytic effects by interacting with the membranes of bacterial cells. Direct analyses of peptide interactions with real cellular membranes are difficult, however, due to the high complexity of physiological membranes. This review summarizes experimental work aiming to understand peptide-membrane interactions and their relationships with the peptides' biological actions using specific model systems. Varied model assemblies have been constructed that generally aim to mimic the fundamental lipid bilayer organization of the membrane. The model systems we will describe include multilamellar and unilamellar vesicles, planar lipid bilayers, lipid monolayers and micelles, and colorimetric biomimetic membranes. The different artificial models have facilitated examination of specific biological or chemical parameters affecting peptide action, for example the effect of membrane lipid composition on peptide affinities and membrane penetration, the relationship between membrane fluidity and peptide interactions, the conformations of active peptides, and other factors. We evaluate the strengths and limitations of the various approaches, and point to future directions in the field.     

Effect of the molecular structure of more common amphiphilic molecules on their interactions with dioleoyl-phosphatidylcholine monolayers

A large number of surface-active or amphiphilic compounds interact with biological membranes and their various models. The surface-active properties of these compounds have been cited as a fundamental factor which determines the tendency of these molecules to bind to, to cross and to perturb the lipid structure of biomembranes and biomembranes models. As well as surface activity, the extent of interaction of a compound with a membrane has been correlated with its lipophilicity. This study shows that the surface activity and lipophilicity of a compound are not the only factors which determine its interaction with a lipid membrane structure. Experiments have been carried out looking at the effect of compounds of different molecular structure on their type of association with self-assembled phospholipid monolayers on mercury electrodes. The paper concludes by demonstrating the properties of different compounds which facilitate their interaction with the supported lipid monolayer.     

Influence of lipidation of GBV-C/HGV NS3 (513-522) and (505-514) peptide sequences on its interaction with mono and bilayers

Two decapeptide fragments of the non-structural hepatitis G NS3 protein (GBV-C/HGV), 513-522 (RGRTGRGRSG) and 505-514 (SAELSMQRRG), as well as their palmitoylated derivatives were synthesized. The physico-chemical properties of the peptides were analyzed in both the absence and presence of the zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), the negative 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) and the positive 1,2-dioeloyl-3-trimethylammonium-propane (DOTAP) lipid monolayers. Based on their high hydrophilic properties, neither parent peptide presented surface activity and their incorporation into lipid monolayers was low. In contrast, their palmitoylated derivatives showed concentration-dependent surface activity and could be inserted into lipid monolayers to varying degrees depending on their sequence. Compression isotherms showed that the presence of palmitoylated peptides in the subphase resulted in a molecular arrangement less condensed than that corresponding to the pure phospholipid. In concordance with the monolayer results, differential scanning calorimetry (DSC) demonstrated that the parent peptides did not have any effect on the thermograms, while the palmitoylated derivatives affected the thermotropic properties of DPPC bilayers.     

Molecular Dynamics Simulation of Small Molecules Interacting with Biological Membranes

Cell membranes protect and compartmentalise cells and their organelles. The semi-permeable nature of these membranes controls the exchange of solutes across their structure. Characterising the interaction of small molecules with biological membranes is critical to understanding of physiological processes, drug action and permeation, and many biotechnological applications. This review provides an overview of how molecular simulations are used to study the interaction of small molecules with biological membranes, with a particular focus on the interactions of water, organic compounds, drugs and short peptides with models of plasma cell membrane and stratum corneum lipid bilayers. This review will not delve on other types of membranes which might have different composition and arrangement, such as thylakoid or mitochondrial membranes. The application of unbiased molecular dynamics simulations and enhanced sampling methods such as umbrella sampling, metadynamics and replica exchange are described using key examples. This review demonstrates how state-of-the-art molecular simulations have been used successfully to describe the mechanism of binding and permeation of small molecules with biological membranes, as well as associated changes to the structure and dynamics of these membranes. The review concludes with an outlook on future directions in this field.     

Patterned supported lipid bilayers and monolayers on poly(dimethylsiloxane)

A simple and practical method for patterning supported lipid bilayers on poly(dimethylsiloxane) is presented. By using electron microscopy grids to laterally control the extent of plasma oxidation, the substrate is partitioned into regions of different hydrophilicities. Addition of vesicles then results in the spontaneous formation of lipid bilayers and monolayers side-by-side on the surface, separated by regions that contain no lipid and/or a region with adhering vesicles. By using millimeter-sized plastic masks we are able to control the formation of these lipid structures on macroscopic patches by simply varying the plasma-cleaning time. For the first time, we are able to influence, in a controlled fashion, the chemical composition of a substrate in such a way that it supports fluid lipid monolayers, rejects lipid adhesion, adsorbs intact lipid vesicles, or supports fluid bilayers.     

Interactions in lipid stabilised foam films

The interaction between lipid bilayers in water has been intensively studied over the last decades. Osmotic stress was applied to evaluate the forces between two approaching lipid bilayers in aqueous solution. The force–distance relation between lipid mono- or bilayers deposited on mica sheets using a surface force apparatus (SFA) was also measured. Lipid stabilised foam films offer another possibility to study the interactions between lipid monolayers. These films can be prepared comparatively easy with very good reproducibility. Foam films consist usually of two adsorbed surfactant monolayers separated by a layer of the aqueous solution from which the film is created. Their thickness can be conveniently measured using microinterferometric techniques. Studies with foam films deliver valuable information on the interactions between lipid membranes and especially their stability and permeability. Presenting inverse black lipid membrane (BLM) foam films supply information about the properties of the lipid self-organisation in bilayers. The present paper summarises results on microscopic lipid stabilised foam films by measuring their thickness and contact angle. Most of the presented results concern foam films prepared from dispersions of the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) and some of its mixtures with the anionic lipid — 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG).     

Mechanisms of the interaction of alpha-helical transmembrane peptides with phospholipid bilayers

The synthetic peptide acetyl-K(2)-G-L(24)-K(2)-A-amide (P(24)) and its analogs have been successfully utilized as models of the hydrophobic transmembrane alpha-helical segments of integral membrane proteins. The central polyleucine region of these peptides was designed to form a maximally stable, very hydrophobic alpha-helix which will partition strongly into the hydrophobic environment of the lipid bilayer core, while the dilysine caps were designed to anchor the ends of these peptides to the polar surface of the lipid bilayer and to inhibit the lateral aggregation of these peptides. Moreover, the normally positively charged N-terminus and the negatively charged C-terminus have both been blocked in order to provide a symmetrical tetracationic peptide, which will more faithfully mimic the transbilayer region of natural membrane proteins and preclude favorable electrostatic interactions. In fact, P(24) adopts a very stable alpha-helical conformation and transbilayer orientation in lipid model membranes. The results of our recent studies of the interaction of this family of alpha-helical transmembrane peptides with phospholipid bilayers are summarized here.     

Physicochemical characterisation of four peptide sequences related to thrombospondin-1B

Thrombospondin-1 (TSP-1) is a protein involved in angiogenesis and tumor metastasis. In a previous study, a tridecapeptide sequence of TSP-1B [KRFKQDGGWSHWG] was synthesized and its biological activity was determined as well as the activity of three related sequences TSPB-(E), TSPB-(S), and TSPB-(Abu)(6). These peptides were tested for activity on the cell growth of three human carcinoma cells lines and only TSPB-(Abu)(6) increased proliferation of MCF7 and HT-29. The main aim of this study was to perform physicochemical measurements, in a comparative way, to determine if the differences in activity could be related to physicochemical properties. Peptides were characterised by HPLC capacity factors, UV, fluorescence, and CD spectra (either in buffer solution or in the presence of lipid vesicles), surface activity, and aggregation. Moreover, the interaction of these peptides with phospholipids was determined through their penetration in monolayers of DPPC, PG, or PS as well as their miscibility in mixed monolayers. Besides, using liposomes as model membranes, the affinity of these peptides for phosphatidylcholine was measured with vesicles labeled with fluorescent markers (TMA-DPH, laurdan, pyrene). Results show that these molecules are highly hydrophilic and their surface activity is low. Mixed monolayers indicate that there is almost no miscibility. Besides, its presence does not modify noticeably the microviscosity of bilayers. Moreover, UV and fluorescence spectra of peptides were not affected by the presence of lipids in the media but CD spectra recorded in TFE/water (1/1) resulted in small changes for TSPB, TSPB-(E), and TSPB-(S) peptides. On the contrary CD spectra of TSPB-(Abu)(6) derivatives were clearly much more sensitive to the polarity of the environment. According to these data the biological activity of peptide with a cyclic aspartimide moiety at position 6 could be related to a specific conformational change in the peptide chain promoted by a hydrophobic membrane-like environment.     

Synthesis and physicochemical study of the laminin active sequence: SIKVAV

The synthesis, physicochemical characterization, and interaction with membrane model systems of a peptide derived from the PA22-2 region of laminin are described. Surface activity studies indicate that this peptide is able to spread at the air-water interface being the maximal spreading pressure 20 mN/m at subphase concentrations around 10 micro M. Besides, these peptide molecules are also able to form stable monolayers. Physicochemical studies concerning the interaction of this peptide with lipids, organized in mono and bilayers, were carried out using Langmuir balance experiments and polarization fluorescence techniques. The peptide penetrates better in monolayers of DPPC than in those of PC and forms condensed mixed monolayers with DPPC. Energies of mixing are small thus indicating that deviations from ideality were almost negligible. Interactions with bilayers were studied through microviscosity changes (DPH and TMA-DPH probes), membrane permeability alterations (CF, NBD-PE/dithionite), and fusion promotion (NBD-PE/Rh-PE, resonance energy transfer). Results indicate that this sequence interacts very softly with bilayers without promoting changes in their organization. These data as well as the lack of interaction with erythrocytes suggest that coating liposomes with this peptide through chemical amide bonds can render stable inmunoliposomes for further biological applications.     

Directed assembly of surface-supported bilayers with transmembrane helices

The lateral assembly of transmembrane (TM) helices gives rise to membrane proteins with complex folds, which play important roles in biochemical processes. Therefore, the assembly of surface-supported bilayers containing TM helices is the first step toward the development of functional biomembrane mimetics. Here we report novel directed assembly of surface-supported lipid bilayers with laterally mobile TM helices. The TM helices were incorporated into lipid monolayers at the air/water interface, and the monolayers were then transferred onto glass substrates using Langmuir-Blodgett (LB) deposition. Finally, bilayers were assembled using lipid vesicle fusion on top of the LB monolayers. The novelty is the incorporation of the peptides into the monolayer at the first step of bilayer assembly, which allows control over the peptide concentration and orientation. The transmembrane orientation of the peptides was confirmed using oriented circular dichroism (OCD), lateral mobility was assessed using fluorescence recovery after photobleaching (FRAP), and diffusion coefficients were determined using a novel boundary profile evolution (BPE) method. The described directed-assembly approach can be used to develop versatile bilayer platforms for studying membrane proteins interactions in native bilayer environments.     

Negative dipole potentials of uncharged langmuir monolayers due to fluorination of the hydrophilic heads

The dipole potential, affecting the structure, functions, and interactions of biomembranes, lipid bilayers, and Langmuir monolayers, is positive toward the hydrocarbon moieties. We show that uncharged Langmuir monolayers of docosyl trifluoroethyl ether (DFEE) exhibit large negative dipole potentials, while the nonfluorinated docosyl ethyl ether (DEE) forms films with positive dipole potentials. Comparison of the Delta V values for these ethers with those of the previously studied(37-39) monolayers of trifluoroethyl ester (TFEB) and ethyl ester of behenic acid (EB) shows that the reversal of the sign of Delta V causes the same change Delta(Delta V) = -706 +/- 16 mV due to fluorination of heads. The Delta V values of both TFEB and EB films differ by -122 +/- 16 mV from those of DFEE and DEE monolayers, respectively, with the same density. Such quantitative coincidence points to a common mechanism of reversal of the sign of the dipole potential for the ether and ester films despite the different structure of their heads. The mechanical properties and phase behaviors of these monolayers show that both fluorinated heads are less hydrated, suggesting that the change of the sign of Delta V could, at least partially, be related to different hydration water structure. The same negative contribution of the carbonyl bond in both TFEB and EB films contrasts with the generally accepted positive contribution of the C(delta+)=O(delta-) bond in condensed Langmuir monolayers of fatty acids, their alcohol esters, glycerides, and phospholipids but concurs with the theoretical analysis of Delta V of stearic acid monolayers. Both results question the literature values of the molecular dipole moments of these substances calculated via summation of bonds and atomic group contributions. Mixed monolayers of DFEE and DEE show smooth monotonic variation of Delta V from +450 to -235 mV, indicating a way for adjustment of the sign and magnitude of the dipole potential at the membrane-water boundary and regulation of such membrane behaviors as binding and translocation rate of hydrophobic ions and ion-carriers, adsorption and penetration of amphiphilic peptides, polarization of hydration water, and short-range repulsion. The interaction of the hydrophobic ions tetraphenylboron TPhB- and tetraphenylphosphonium TPhP+ with DFEE and DEE monolayers qualitatively follows the theory of binding of such ions to lipid bilayers, but the shifts Delta(Delta V) from the values obtained on water are much smaller than those for DPPC monolayers. This difference seems to be due to the solid (polycrystalline) character of the DFEE and DEE films that hampers the penetration of TPhB- and TPhP+ in the monolayers and reduces the attractive interaction with the hydrophobic moiety. This conclusion orients the future synthesis of amphiphiles with fluorinated heads to those which could form liquid-expanded Langmuir monolayers.     

Phase behavior and the partitioning of caveolin-1 scaffolding domain peptides in model lipid bilayers

The membrane binding and model lipid raft interaction of synthetic peptides derived from the caveolin scaffolding domain (CSD) of the protein caveolin-1 have been investigated. CSD peptides bind preferentially to liquid-disordered domains in model lipid bilayers composed of cholesterol and an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and brain sphingomyelin. Three caveolin-1 peptides were studied: the scaffolding domain (residues 83-101), a water-insoluble construct containing residues 89-101, and a water-soluble construct containing residues 89-101. Confocal and fluorescence microscopy investigation shows that the caveolin-1 peptides bind to the more fluid cholesterol-poor phase. The binding of the water-soluble peptide to lipid bilayers was measured using fluorescence correlation spectroscopy (FCS). We measured molar partition coefficients of 10(4) M(-1) between the soluble peptide and phase-separated lipid bilayers and 10(3) M(-1) between the soluble peptide and bilayers with a single liquid phase. Partial phase diagrams for our phase-separating lipid mixture with added caveolin-1 peptides were measured using fluorescence microscopy. The water-soluble peptide did not change the phase morphology or the miscibility transition in giant unilamellar vesicles (GUVs); however, the water-insoluble and full-length CSD peptides lowered the liquid-liquid melting temperature.     

Amphiphilic peptides as models for protein-membrane interactions: interfacial behaviour of sequential Lys- and Leu-based peptides and their penetration into lipid monolayers

Synthetic peptides constructed with doublets of hydrophobic residues tandemly repeated with doublets of positively charged residues, (Leu-Lys-Lys-Leu)_n, were used as models for the study of protein-membrane interactions. Their behaviour has been compared with that of their strictly alternating iso peptides, (Leu-Lys)_n. Both peptides present a random coil structure in pure water. In saline solutions, (Leu-Lys-Lys-Leu)_n peptides adopt an α-helical structure whereas (Leu-Lys)_n transit into a β-sheet structure. These peptides form multilayer assemblies on a pure water subphase but they are organized in monomolecular films on a saline aqueous subphase. The stability of these films increases with the peptide length. Structured peptides (α helices and β sheets) penetrate readily into lipid monolayers, whereas the penetration of unordered peptides is very slow. We have not observed any significant difference between the behaviour of a helices and β-sheet structures.     

Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy

There is substantial scientific and practical interest in engineering supported lipid bilayers with asymmetric lipid distributions as models for biological cell membranes. In principle, it should be possible to make asymmetric supported lipid bilayers by either the Langmuir-Blodgett/Schafer (LB/LS) or Langmuir-Blodgett/vesicle fusion (LB/VF) techniques (Kalb et al. Biochim. Biophys. Acta 1992, 1103, 307-316). However, the retention of asymmetry in biologically relevant lipid bilayers has never been experimentally examined in any of these systems. In the present work, we developed a technique that is based on fluorescence interference contrast (FLIC) microscopy to measure lipid asymmetry in supported bilayers. We compared the final degree of lipid asymmetry in LB/LS and LB/VF bilayers with and without cholesterol in liquid-ordered (l(o)) and liquid-disordered (l(d)) phases. Of five different fluorescent lipid probes that were examined, 1,2-dipalmitoyl-phosphatidylethanolamine-N-[lissamine rhodamine B] was the best for studying supported bilayers of complex composition and phase by FLIC microscopy. An asymmetrically labeled bilayer made by the LB/LS method was found to be at best 70-80% asymmetric once completed. In LB/LS bilayers of either l(o) or l(d) phase, cholesterol increased the degree of lipid mixing between the opposing monolayers. The use of a tethered polymer support for the initial monolayer did not improve lipid asymmetry in the resulting bilayer. However, asymmetric LB/VF bilayers retained nearly 100% asymmetric label, with or without the use of a tethered polymer support. Finally, lipid mixing across the center of LB/LS bilayers was found to have drastic effects on the appearance of l(d)-l(o) phase coexistence as shown by epifluorescence microscopy.     

Long-range Lennard-Jones and electrostatic interactions in interfaces: application of the isotropic periodic sum method

Molecular dynamics (MD) simulations of heptane/vapor, hexadecane/vapor, water/vapor, hexadecane/water, and dipalmitoylphosphatidylcholine (DPPC) bilayers and monolayers are analyzed to determine the accuracy of treating long-range interactions in interfaces with the isotropic periodic sum (IPS) method. The method and cutoff (rc) dependences of surface tensions, density profiles, water dipole orientation, and electrostatic potential profiles are used as metrics. The water/vapor, heptane/vapor, and hexadecane/vapor interfaces are accurately and efficiently calculated with 2D IPS (rc=10 A). It is demonstrated that 3D IPS is not practical for any of the interfacial systems studied. However, the hybrid method PME/IPS [Particle Mesh Ewald for electrostatics and 3D IPS for Lennard-Jones (LJ) interactions] provides an efficient way to include both types of long-range forces in simulations of large liquid/vacuum and all liquid/liquid interfaces, including lipid monolayers and bilayers. A previously published pressure-based long-range LJ correction yields results similar to those of PME/IPS for liquid/liquid interfaces. The contributions to surface tension of LJ terms arising from interactions beyond 10 A range from 13 dyn/cm for the hexadecane/vapor interface to approximately 3 dyn/cm for hexadecane/water and DPPC bilayers and monolayers. Surface tensions of alkane/vapor, hexadecane/water, and DPPC monolayers based on the CHARMM lipid force fields agree very well with experiment, whereas surface tensions of the TIP3P and TIP4P-Ew water models underestimate experiment by 16 and 11 dyn/cm, respectively. Dipole potential drops (DeltaPsi) are less sensitive to long-range LJ interactions than surface tensions. However, DeltaPsi for the DPPC bilayer (845+/-3 mV proceeding from water to lipid) and water (547+/-2 mV for TIP4P-Ew and 521+/-3 mV for TIP3P) overestimate experiment by factors of 3 and 5, respectively, and represent expected deficiencies in nonpolarizable force fields.     

Study of the interaction of human defensins with cell membrane models: relationships between structure and biological activity

The HNP-1, HNP-2, and HNP-3 defensins are human antimicrobial peptides produced in response to microbial invasion. Their properties are distinct, with a more potent action for HNP-3. In this study, the relationship between their structural dissimilarities and their different microbial actions was evaluated by molecular dynamics simulation. Structural determinants related to their intra- and intermolecular interactions were defined for each HNP using a simplified membrane model consisting of a water/n-hexane interface. The hydrophobic portion of the HNPs promotes their diffusion to the interface with a concomitant, slight change in the structure induced by the intermolecular electrostatic interactions between the HPN molecules and the interface. As a consequence, different orientations are probably adopted by the HNPs at the interface, which may explain their different actions. The interaction of HNP-1 and HNP-2 with the surfaces was also studied using Langmuir monolayers as a biomimetic system. It was found that peptides adsorb rapidly at n-hexane/water interfaces as well as at phospholipid Langmuir monolayers but not at the air/liquid interface. This reveals that the presence of an organic phase is required for the exposure of the hydrophobic groups of the peptides. In addition, adsorption kinetics and surface pressure-area isotherms for Langmuir monolayers suggested that the lipid-peptide interaction is strongly influenced by the monolayer electrical charge and packing, depending also on the HPN structure. This study supports a model in which defensins, acting in a dimeric form, are able to disrupt membranes. The model also shows that the individual structures of the HNPs are responsible for their different actions on microbes.     

An electrochemical study into the interaction between complement-derived peptides and DOPC mono- and bilayers

Electrochemical methods employing the hanging mercury drop electrode were used to study the interaction between variants of the complement-derived antimicrobial peptide CNY21 (CNYITELRRQH ARASHLGLAR) and dioleoyl phosphatidylcholine (DOPC) monolayers. Capacitance potential and impedance measurements showed that the CNY21 analogues investigated interact with DOPC monolayers coating the mercury drop. Increasing the peptide hydrophobicity by substituting the two histidine residues with leucine resulted in a deeper peptide penetration into the hydrophobic region of the DOPC monolayer, indicated by an increase in the dielectric constant of the lipid monolayer (Deltaepsilon = 2.0 after 15 min interaction). Increasing the peptide net charge from +3 to +5 by replacing the histidines by lysines, on the other hand, arrests the peptide in the lipid head group region. Reduction of electroactive ions (Tl+, Pb2+, Cd2+, and Eu3+) at the monolayer-coated electrode was employed to further characterize the types of defects induced by the peptides. All peptides studied permeabilize the monolayer to Tl+ to an appreciable extent, but this effect is more pronounced for the more hydrophobic peptide (CNY21L), which also allows penetration of larger ions and ions of higher valency. The results for the various ions indicate that charge repulsion rather than ion size is the determining factor for cation penetration through peptide-induced defects in the DOPC monolayer. The effects obtained for monolayers were compared to results obtained with bilayers from liposome leakage and circular dichroism studies for unilamellar DOPC vesicles, and in situ ellipsometry for supported DOPC bilayers. Trends in peptide-induced liposome leakage were similar to peptide effects on electrochemical impedance and permeability of electroactive ions for the monolayer system, demonstrating that formation of transmembrane pores alone does not constitute the mechanism of action for the peptides investigated. Instead, our results point to the importance of local packing defects in the lipid membrane in close proximity to the adsorbed peptide molecules.     

Microcontact printing for creation of patterned lipid bilayers on tetraethylene glycol self-assembled monolayers

Supported lipid bilayers (SLBs) formed on many different substrates have been widely used in the study of lipid bilayers. However, most SLBs suffer from inhomogeneities due to interactions between the lipid bilayer and the substrate. In order to avoid this problem, we have used microcontact printing to create patterned SLBs on top of ethylene-glycol-terminated self-assembled monolayers (SAMs). Glycol-terminated SAMs have previously been shown to resist absorbance of biomolecules including lipid vesicles. In our system, patterned lipid bilayer regions are separated by lipid monolayers, which form over the patterned hexadecanethiol portions of the surface. Furthermore, we demonstrate that α-hemolysin, a large transmembrane protein, inserts preferentially into the lipid bilayer regions of the substrate.